Junqin Chen

Intracellular Shuttling and Mitochondrial Function of Thioredoxin-interacting Protein

The thioredoxin-interacting protein TXNIP is a ubiquitously expressed redox protein that promotes apoptosis. Recently, we found that TXNIP deficiency protects against type 1 and 2 diabetes by inhibiting beta cell apoptosis and maintaining pancreatic beta cell mass, indicating that TXNIP plays a key role in beta cell biology. However, very little is known about the intracellular localization and function of TXNIP, and although TXNIP has been thought to be a cytoplasmic protein, our immunohistochemistry studies in beta cells surprisingly revealed a nuclear TXNIP localization, suggesting that TXNIP may shuttle within the cell. Using immunohistochemistry/confocal imaging and cell fractionation/co-immunoprecipitation, we found that, under physiological conditions, TXNIP is localized primarily in the nucleus of pancreatic beta cells, whereas oxidative stress leads to TXNIP shuttling into the mitochondria. In mitochondria, TXNIP binds to and oxidizes Trx2, thereby reducing Trx2 binding to ASK1 and allowing for ASK1 phosphorylation/activation, resulting in induction of the mitochondrial pathway of apoptosis with cytochrome c release and caspase-3 cleavage. TXNIP overexpression and Trx2 (but not cytosolic Trx1) silencing mimic these effects. Thus, we discovered that TXNIP shuttles between subcellular compartments in response to oxidative stress and identified a novel redox-sensitive mitochondrial TXNIP-Trx2-ASK1 signaling cascade.

Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes

Pancreatic beta‐cell loss through apoptosis represents a key factor in the pathogenesis of diabetes;however, no effective approaches to block this process and preserve endogenous beta‐cell mass are currently available. To study the role of thioredoxin‐interacting protein (TXNIP), a proapoptotic beta‐cell factor we recently identified, we used HcB‐19 (TXNIP nonsense mutation) and beta‐cell‐specific TXNIP knockout (bTKO) mice. Interestingly, HcB‐19 mice demonstrate increased adiposity, but have lower blood glucose levels and increased pancreatic beta‐cell mass (as assessed by morphometry). Moreover, HcB‐19 mice are resistant to streptozotocin‐induced diabetes. When intercrossed with obese, insulin‐resistant, and diabetic mice, double‐mutant BTBRlepob/obtxniphcb/hcb are even more obese, but are protected against diabetes and beta‐cell apoptosis, resulting in a 3‐fold increase in beta‐cell mass. Beta‐cell‐specific TXNIP deletion also enhanced beta‐cell mass (P< 0.005) and protected against diabetes, and terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) revealed a ~50‐fold reduction in beta‐cell apoptosis in streptozotocin‐treated bTKO mice. We further discovered that TXNIP deficiency induces Akt/Bcl‐xL signaling and inhibits mitochondrial beta‐cell death, suggesting that these mechanisms may mediate the beta‐cell protective effects of TXNIP deficiency. These results suggest that lowering beta‐cell TXNIP expression could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta‐cell survival.—Chen, J., Hui, S. T., Couto, F. M., Mungrue, I. N., Davis, D. B., Attie, A. D., Lusis, A. J., Davis, R. A., Shalev, A. Thioredoxin‐interacting protein deficiency induces Akt/ Bcl‐xL signaling and pancreatic beta‐cell mass and protects against diabetes. FASEB J. 22, 3581–3594 (2008)

Glucose-stimulated Expression of Txnip Is Mediated by Carbohydrate Response Element-binding Protein, p300, and Histone H4 Acetylation in Pancreatic Beta Cells

Recently, we identified Txnip (thioredoxin-interacting protein) as a mediator of glucotoxic beta cell death and discovered that lack of Txnip protects against streptozotocin- and obesity-induced diabetes by preventing beta cell apoptosis and preserving endogenous beta cell mass. Txnip has therefore become an attractive target for diabetes therapy, but although we have found that txnip transcription is highly induced by glucose through a unique carbohydrate response element, the factors controlling this effect have remained unknown. Using transient transfection experiments, we now show that overexpression of the carbohydrate response element-binding protein (ChREBP) transactivates the txnip promoter, whereas ChREBP knockdown by small interfering RNA completely blunts glucose-induced txnip transcription. Moreover, chromatin immunoprecipitation demonstrated that glucose leads to a dose- and time-dependent recruitment of ChREBP to the txnip promoter in vivo in INS-1 beta cells as well as human islets. Furthermore, we found that the co-activator and histone acetyltransferase p300 co-immunoprecipitates with ChREBP and also binds to the txnip promoter in response to glucose. Interestingly, this is associated with specific acetylation of histone H4 and recruitment of RNA polymerase II as measured by chromatin immunoprecipitation. Thus, with this study we have identified ChREBP as the transcription factor that mediates glucose-induced txnip expression in human islets and INS-1 beta cells and have characterized the chromatin modification associated with glucose-induced txnip transcription. In addition, the results reveal for the first time that ChREBP interacts with p300. This may explain how ChREBP induces H4 acetylation and sheds new light on glucose-mediated regulation of chromatin structure and transcription.

Thioredoxin-interacting protein regulates insulin transcription through microRNA-204

Beta-cell dysfunction and impaired insulin production are hallmarks of diabetes, but despite the growing diabetes epidemic, the molecular mechanisms underlying this disease have remained unclear. We identified thioredoxin-interacting protein (TXNIP), a cellular redox regulator, as a crucial factor in beta-cell biology and show that beta-cell TXNIP is upregulated in diabetes, whereas TXNIP deficiency protects against diabetes by preventing beta-cell apoptosis. Here we show that TXNIP and diabetes induce beta-cell expression of a specific microRNA, miR-204, which in turn blocks insulin production by directly targeting and downregulating MAFA, a known insulin transcription factor. In particular, we first discovered the regulation of miR-204 by TXNIP by microarray analysis, followed by validation studies in INS-1 beta cells, islets of Txnip-deficient mice, diabetic mouse models and primary human islets. We then further found that TXNIP induces miR-204 by inhibiting the activity of signal transducer and activator of transcription 3 (STAT3), a transcription factor that is involved in miR-204 regulation. We also identified MAFA as a target that is downregulated by miR-204. Taken together, our results demonstrate that TXNIP controls microRNA expression and insulin production and that miR-204 is involved in beta-cell function. The newly identified TXNIP–miR-204–MAFA–insulin pathway may contribute to diabetes progression and provides new insight into TXNIP function and microRNA biology in health and disease.

Lack of TXNIP Protects Against Mitochondria-Mediated Apoptosis but Not Against Fatty Acid–Induced ER Stress–Mediated β-Cell Death

OBJECTIVE We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced β-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects β-cells against lipoapoptosis. RESARCH DESIGN AND METHODS To determine the effects of fatty acids on β-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). RESULTS Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced β-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. CONCLUSIONS Our results demonstrate that unlike glucose, fatty acids do not induce β-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying β-cell glucotoxicity, whereas it has very few protective effects against ER stress–mediated lipoapoptosis.

Diabetes induces and calcium channel blockers prevent cardiac expression of proapoptotic thioredoxin-interacting protein

Cardiomyocyte apoptosis is a critical process in the pathogenesis of ischemic and diabetic cardiomyopathy, but the mechanisms are not fully understood. Thioredoxin-interacting protein (TXNIP) has recently been shown to have deleterious effects in the cardiovascular system and we therefore investigated whether it may also play a role in diabetes-associated cardiomyocyte apoptosis. In fact, TXNIP expression was increased in H9C2 cardiomyocytes incubated at high glucose, and cardiac expression of TXNIP and cleaved caspase-3 were also elevated in vivo in streptozotocin- and obesity-induced diabetic mice. Together, these findings not only suggest that TXNIP is involved in diabetic cardiomyopathy but also that it may represent a novel therapeutic target. Surprisingly, testing putative TXNIP modulators revealed that calcium channel blockers reduce cardiomyocyte TXNIP transcription and protein levels in a dose-dependent manner. Oral administration of verapamil for 3 wk also reduced cardiac TXNIP expression in mice even in the face of severe diabetes, and these reduced TXNIP levels were associated with decreased apoptosis. To determine whether lack of TXNIP can mimic the verapamil-induced decrease in apoptosis, we used TXNIP-deficient HcB-19 mice, harboring a natural nonsense mutation in the TXNIP gene. Interestingly, we found significantly reduced cleaved caspase-3 levels in HcB-19 hearts, suggesting that TXNIP plays a critical role in cardiac apoptosis and that the verapamil effects were mediated by TXNIP reduction. Thus our results suggest that TXNIP reduction is a powerful target to enhance cardiomyocyte survival and that agents such as calcium channel blockers may be useful in trying to achieve this goal and prevent diabetic cardiomyopathy.

Preventing β-Cell Loss and Diabetes With Calcium Channel Blockers

Although loss of functional β-cell mass is a hallmark of diabetes, no treatment approaches that halt this process are currently available. We recently identified thioredoxin-interacting protein (TXNIP) as an attractive target in this regard. Glucose and diabetes upregulate β-cell TXNIP expression, and TXNIP overexpression induces β-cell apoptosis. In contrast, genetic ablation of TXNIP promotes endogenous β-cell survival and prevents streptozotocin (STZ)- and obesity-induced diabetes. Finding an oral medication that could inhibit β-cell TXNIP expression would therefore represent a major breakthrough. We were surprised to discover that calcium channel blockers inhibited TXNIP expression in INS-1 cells and human islets and that orally administered verapamil reduced TXNIP expression and β-cell apoptosis, enhanced endogenous insulin levels, and rescued mice from STZ-induced diabetes. Verapamil also promoted β-cell survival and improved glucose homeostasis and insulin sensitivity in BTBR ob/ob mice. Our data further suggest that this verapamil-mediated TXNIP repression is conferred by reduction of intracellular calcium, inhibition of calcineurin signaling, and nuclear exclusion and decreased binding of carbohydrate response element–binding protein to the E-box repeat in the TXNIP promoter. Thus, for the first time, we have identified an oral medication that can inhibit proapoptotic β-cell TXNIP expression, enhance β-cell survival and function, and prevent and even improve overt diabetes.

MicroRNA-200 Is Induced by Thioredoxin-interacting Protein and Regulates Zeb1 Protein Signaling and Beta Cell Apoptosis

Background: Beta cell apoptosis is a key factor in diabetes, but the mechanisms are not well understood. Results: Beta cell miR-200 is induced by thioredoxin-interacting protein (TXNIP) and diabetes, directly targets Zeb1, up-regulates E-cadherin, and promotes apoptosis. Conclusion: This novel TXNIP/miR-200/Zeb1/E-cadherin signaling pathway links miR-200 to beta cell apoptosis, control of epithelial-mesenchymal transition, and diabetes. Significance: The results provide new insight into microRNA biology and the regulation of beta cell apoptosis. Small noncoding microRNAs have emerged as important regulators of cellular processes, but their role in pancreatic beta cells has only started to be elucidated. Loss of pancreatic beta cells is a key factor in the pathogenesis of diabetes, and we have demonstrated that beta cell expression of thioredoxin-interacting protein (TXNIP) is increased in diabetes and causes beta cell apoptosis, whereas TXNIP deficiency is protective against diabetes. Recently, we found that TXNIP also impairs beta cell function by inducing microRNA (miR)-204. Interestingly, using INS-1 beta cells and primary islets, we have now discovered that expression of another microRNA, miR-200, is induced by TXNIP and by diabetes. Furthermore, we found that miR-200 targeted and decreased Zeb1 (zinc finger E-box-binding homeobox 1) and promoted beta cell apoptosis as measured by cleaved caspase-3 levels, Bax/Bcl2 ratio, and TUNEL. In addition, Zeb1 knockdown mimicked the miR-200 effects on beta cell apoptosis, suggesting that Zeb1 plays an important role in mediating miR-200 effects. Moreover, miR-200 increased beta cell expression of the epithelial marker E-cadherin, consistent with inhibition of epithelial-mesenchymal transition, a process thought to be involved in beta cell expansion. Thus, we have identified a novel TXNIP/miR-200/Zeb1/E-cadherin signaling pathway that, for the first time, links miR-200 to beta cell apoptosis and diabetes and also beta cell TXNIP to epithelial-mesenchymal transition. In addition, our results shed new light on the regulation and function of miR-200 in beta cells and show that TXNIP-induced microRNAs control various processes of beta cell biology.

FOXO1 Competes with Carbohydrate Response Element-binding Protein (ChREBP) and Inhibits Thioredoxin-interacting Protein (TXNIP) Transcription in Pancreatic Beta Cells

Background: Control of thioredoxin-interacting protein (TXNIP) expression is critical for pancreatic beta cell survival. Results: FOXO1 binds to the TXNIP promoter; blocks ChREBP occupancy, and inhibits glucose-induced beta cell TXNIP transcription. Conclusion: FOXO1 controls glucose-induced gene expression by competing with ChREBP at target promoters, e.g. TXNIP and L-PK. Significance: This represents a novel gene regulatory mechanism and is the first demonstration of FOXO1-ChREBP cross-talk. Thioredoxin-interacting protein (TXNIP) has emerged as an important factor in pancreatic beta cell biology, and tight regulation of TXNIP levels is necessary for beta cell survival. However, the mechanisms regulating TXNIP expression have only started to be elucidated. The forkhead boxO1 transcription factor (FOXO1) has been reported to up-regulate TXNIP expression in neurons and endothelial cells but to down-regulate TXNIP in liver, and the effects on beta cells have remained unknown. We now have found that FOXO1 binds to the TXNIP promoter in vivo in human islets and INS-1 beta cells and significantly decreases TXNIP expression. TXNIP promoter deletion analyses revealed that an E-box motif conferring carbohydrate response element-binding protein (ChREBP)-mediated, glucose-induced TXNIP expression is necessary and sufficient for this effect, and electromobility shift assays confirmed FOXO1 binding to this site. Moreover, FOXO1 blocked glucose-induced TXNIP expression and reduced glucose-induced ChREBP binding at the TXNIP promoter without affecting ChREBP expression or nuclear localization, suggesting that FOXO1 may compete with ChREBP for binding to the TXNIP promoter. In fact, a FOXO1 DNA-binding mutant (FOXO1-H215R) failed to inhibit TXNIP transcription, and the effects were not restricted to TXNIP as FOXO1 also inhibited transcription of other ChREBP target genes such as liver pyruvate kinase. Together, these results demonstrate that FOXO1 inhibits beta cell TXNIP transcription and suggest that FOXO1 confers this inhibition by interfering with ChREBP DNA binding at target gene promoters. Our findings thereby reveal a novel gene regulatory mechanism and a previously unappreciated cross-talk between FOXO1 and ChREBP, two major metabolic signaling pathways.

Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2.

Background: Islet amyloid polypeptide (IAPP) plays an important role in beta-cell biology, but its regulation is not fully understood. Results: Thioredoxin-interacting protein (TXNIP) induces IAPP by inhibiting miR-124a and promoting FoxA2-mediated transcription. Conclusion: The critical beta-cell signaling pathways of TXNIP and IAPP are linked. Significance: Identification of this novel TXNIP/miR-124a/FoxA2/IAPP signaling pathway provides new insight into an important aspect of transcriptional regulation and beta-cell biology. Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.